The present invention relates to recombinant vaccinia virus derived from the vaccinia virus Ankara (MVA) encoding and capable of expressing the E2 gene of Bovine papillomavirus, and the use of such recombinant in the treatment of lesions caused by papillomaviruses.
Cervical Carcinoma
In man neoplasic transformation has been linked to the presence of papillomaviruses. They can induce diseases, from warts up to condilomas, and lesions, which can progress to malignant neoplasias. Approximately one million people get infected with papilloma virus every year, and in developing countries 50,000 women die annually due to cervical cancer.
In humans, it is well known that cervical cancer correlates with the presence of human papillomavirus (HPVS) particularly types 16, 18, 31, 33 and 353,4,5,6,7,8,9,10,11. In contrast, HPV types 6, 11, 42, 43 are found only in the anogenital tract and are not associated with cervical cancer3, 7, 12, 13.
Cervical carcinoma is the second most common cancer among women worldwide. More than 95% of all cervical carcinomas contain DNA of some papillomavirus (HPV)3 with types 16 or 18 accounting for about 50% and 30% of all cases, respectively.
Most benign lesions (papillomas) generally regress on their own, but in some cases, some of them undergo malignant transformation. Papillomavirus are also found infecting a wide variety of vertebrates, and it has been described that they produce tumors in these animals as well4,5,6,7,14.
Normally human papillomavirus infects and replicates in the form of an unintegrated circular episome in keratinocytes of genital mucosa and perigenital skin. The papillomavirus E2 and E1 gene products regulate viral DNA replication. The E2 gene product can also activate or repress transcription of different HPVs promoters15,16. In particular, the papillomavirus protein E2 is known to down regulate the P105 promoter, from HPV18, which controls transcription of the E6 and E7 oncogenes, which are expressed in a variety of cervical human tumors. The best studied HPVs, types 16 and 18 persist extracromosomally in precancerous lesions, but frequently get integrated into the cellular genome of cancer cells16. This event makes the papillomavirus E2 gene disrupted or inactivated leading to a derepression of the E6 and E7 oncogenes. Expression of these oncogenes appears to be a critical step in the maintenance of the transformed stage and progression to invasive carcinoma. This mechanism, however, is not necessarily the only one operating to induce the malignant stage, as suggested by recent studies where HPV integration was not detected in at least 30% of the analyzed cancers13,17.
Since cervical cancer is a serious health problem and because there is a strong dependence with the infection by HPV, it is thought that the induction of a protective stage against these viruses would help in preventing the appearance of cervical tumors. Based on this idea, different strategies to develop a safe vaccine or immunotherapeutic agent against cervical cancer have been tried. Other methods, such as radiotherapy and chemotherapy have, of course been used to reduce papillomas, and cancer tumors. However, these methods work only efficiently during the first stages of tumor development. At later times it becomes very difficult to treat cervical tumors because of the tumor size and the side effects that anti-cancer drugs may have.
Because of the strong relationship between HPV infection and cervical cancer, many approaches have been tried in an effort to develop a successful therapy for cervical cancer. It has been shown that antisense oligonucleotides from partial or complete full-end clones of E6 and E7 genes are able to inhibit tumor cell growth in vitro and in vivo18,19,20 by down regulating the E6 and E7 oncogenes. Cloned antigens, recognized by therapeutic antitumor lymphocytes (TAA), such as the B7-1 or B7-2 molecules, have been used in the construction of recombinant anticancer vaccines21.
Using a recombinant vaccinia virus expressing the B7-1 molecule, it was found that the number of pulmonary metastases decreased in mice bearing tumor21. These molecules interact wit the T lymphocyte ligands CD28 and CTLA-4, and initiate a cascade of effects mediated, at least in part by up-regulation of interleukin 2 production22,23,24.
The findings that the E2 protein of papillomavirus is capable of promoting cell growth arrest and stopping cell proliferation by inducing apoptosis of human cancer cells15,25,26, and that immunized animals with recombinant E2 proteins are capable of inducing tumor regression and decreasing the number of new papilloma foci formed27, have lead to a novel approach for cervical cancer therapy. Namely, the introduction of the E2 gene into vaccinia virus was to efficiently direct the E2 protein against HPV tumors.
Inserting an antigen in a vaccinia virus (poxyirus) increases the expression of this molecule in the infected cell, therefore stimulating more efficiently the immune system28,29. The purpose of using different antigens expressed in vaccinia virus is to try to enhance the immune response against these specific antigens. For these reasons it is thought that recombinant poxvirus are excellent candidates for a new type of vaccine and also for new therapeutic strategies.
Knowing these difficulties to treat cervical cancer it was decided to use an attenuated vaccinia virus known as Modified Vaccinia Ankara (MVA). This virus was developed and tested as a safe smallpox vaccine30. It was also found to be a virulent for normal or immunosuppressed animals, without side-effects in 120,000 humans inoculated for priming vaccination30,31,32. One reason that makes the MVA very safe is that viral expression and recombinant mechanisms are impaired for this virus. It has also been demonstrated that MVA is an excellent vector for expressing foreign genes, such as the Escherichia coli Lac Z or the page T7 polymerase33,34 in infected cells. Moreover, MVA is capable of infecting most, if not all, the human cell lines tested up to now. Because of these characteristics, at the present time, the most successful strategy for vaccine development involves the use of vaccinia virus vectors.
This approach has already been used to protect animals against polyoma virus (PY) infection35. In these reports, it was demonstrated that treatment of rats with live recombinant vaccinia viruses expressing tumor-specific antigens from PY could prevent cognate tumor development and in some cases could also induce regression of preexisting tumors. Also an MVA recombinant virus expressing the haemaglutinin and nucleoprotein genes (HA and NP, respectively) of influenza virus (A/PR/8/34HA) was found to fully protect mice against a lethal influenza virus challenge36.
According to the invention, a DNA sequence which codes for the E2 gene of Bovine papillomavirus is introduced using DNA recombination techniques within a naturally occurring DNA sequence of the MVA genome which is non-essential for virus replication. This recombinant virus will be helpful for the therapy of lesions induced by papillomaviruses.
The present invention is to provide a recombinant MVA virus that expresses a papillomavirus antigen and is used as a therapeutic method to treat lesions generated by papillomaviruses.
The scope of this invention covers the necessary methods and the technology to express a specific gene in a viral expression vector (vaccinia virus strain MVA) specifically in a recombinant viral vector that carries the E2 gene of the bovine papilloma virus. This recombinant vector is capable of efficiently expressing the E2 gene inside any mammalian cell as well as in chicken fibroblast cells.
The recombinant MVA E2 virus as designed basically to be used in the therapeutic treatment of early/late lesions that are produced by infections with papilloma virus capable of generating tumors. This virus was mainly designed to inoculate directly into lesions produced by human papilloma virus.
The MVA E2 virus can be used in the elimination of pre-cancer lesions and has potential use in the elimination of viruses that provoke these lesions. The MVA E2 virus can be used in the treatment of lesions produced by any type of oncogene virus. The MVA E2 virus can be used in the regression of pre-cancerous lesions. The MVA E2 virus has the capacity to induce regression of cancer tumors.
A recombinant MVA containing and capable of expressing a DNA sequence encoding a Bovine papillomavirus protein.
A recombinant MVA as above containing and capable of expressing DNA sequence of the E2 gene of Bovine papillomavirus.
DNA sequences are inserted at the site of naturally occurring deletions within MVA genome.
A recombinant MVA according as above, wherein the DNA sequences encoding antigen is under transcriptional control of the vaccinia virus early/late promoter P7.5.
A recombinant MVA as above and a pharmaceutically acceptable carrier or diluent.
A recombinant MVA as above containing and MVA encoding a Bovine papillomavirus antigen, and a pharmaceutically acceptable carrier or diluent.
A method for the treatment of all type of lesions generated by papillomavirus infections comprising administering to a living animal body, including a human, in need thereof a therapeutically effective amount of a recombinant MVA as above.
A method of treating a tumor lesion as above, comprising administering directly to the tumor a recombinant MVA expressing the E2 gene of Bovine papillomavirus.
A method of inducing anti-tumor antibodies in a tumor-bearing animal, comprising administering a recombinant MVA as above.
The present invention relates to recombinant MVA that contain the E2 gene of bovine papillomavirus suitable in a physiologically acceptable form for the therapy of lesions caused by papillomaviruses. The invention also relates to methods for the preparation of such recombinant MVA virus.
Because it has been reported that the tumor growth can be reduced if sufficient amount of E2 protein is inside the cells38, and recent experiments have showed that MVA recombinant virus carrying the E2 gene of bovine papillomarvirus is capable of stopping human tumor growth generated in nude mice by down regulating the expression of the E6 and E7 oncogenes and also generated apoptosis through P53 pathway. In this patent it is explored the therapeutic potential of recombinant vaccinia expressing the E2 gene of papillomavirus (MVA E2) in the treatment of an established tumor.
It was found in the MVA E2-treated animals that tumors stop growing, while in animals treated with MVA or PBS tumors continue (to grow. The MVA E2-treated animals presented complete tumor regression and were free of tumors for more than a year. A strong immune response was generated against vaccinia and E2 papilloma proteins as indicated by the presence of antibodies against these molecules. No evidence for complement-mediated or lymphocyte-mediated cytotoxicity of tumor cells was found. However, in tumor bearing rabbits treated with the MVA E2 virus, we also found large amounts of anti-tumor antibodies. These antibodies were capable of stimulating macrophages for efficient killing of tumor cells. These data strongly suggest that MVA E2-treated rabbits produce anti-tumor antibodies that stimulate macrophage antibody-dependent tumor cytotoxicity39.
Based on this strategy, we decided to investigate the use of a recombinant MVA virus for therapeutic treatment of cervical cancer. Because the E2 protein represses expression of E6 and E7 proteins, and recent experiments showed that the E2 protein can also induce P53-mediated apoptosis in HeLa cells, and cell growth arrest independently of the transcriptional repression of the endogenous viral E6 and E7 genes11,15,26, we decided to construct a MVA recombinant virus by inserting the E2 gene into the MVA genome. The new recombinant virus was named MVA E2 and its effects over human tumor growth in immunosuppressed animals were investigated.
In our experimental work, it was found that treatment with the recombinant MVA E2 virus resulted in tumor growth arrest in tumor-bearing mice. In contrast, only a slight inhibition of tumor growth was observed in animals inoculated with the parenteral MVA virus, and in control animals, just inoculated with PBS, the tumors continued to grow indefinitely. In addition, MVA E2 was capable of inducing strong tumor regression in tumor-bearing rabbits up to complete disappearance of the malignant tumor.
These data indicate that the recombinant MVA E2 has the capacity of inducing tumor elimination in animals and therefore it is a potential new therapeutic agent for cancer treatment38.
Extract of the Invention:
A method is described to produce an expression viral vector with the recombinant vaccinia virus with the capacity to express the E2 gene of bovine papilloma virus in mammalian cells and in chicken embryo fibroblasts. The method includes the construction of the recombinant transfer vector through the insertion of a fragment of DNA (promoter of the vaccinia virus P7.5 E/L) and with the later insertion of a selected gene (E2 gene of bovine papilloma virus), so the new cloning vector is under the transcriptional control of promoter P7.5 E/L. The recombinant transfer vector is put in contact with DNA of the vaccinia virus to carry-out the recombination and the incorporation of the selected gene into the genome of the vaccinia virus. The recombinant vaccinia virus that results from this construction is used then to infect mammalian cells or chicken fibroblast cells in culture and, as a product of the infection, the protein of E2 gene is produced because it was selected and incorporated into the DNA of the vaccinia virus.
The recombinant virus produced as described above is named MVA E2, it is purified until it reaches 97% of purity, and then can be used in the treatment of early lesions as well as in preexisting tumors that have originated by any type of oncogenic papilloma virus. The invention of this recombinant virus will facilitate the therapeutic treatment of pre-cancer and cancer lesions as well as of present papillomas.
It has been proven that the recombinant virus MVA E2 can reduce and eliminate tumors, both in immunocompetent animals as well in those that do not have present an immune system (mice and rabbits). The MVA E2 virus has a high potential to eradicate oncogenic papilloma viruses and this is mainly due to the fact that recombinant virus MVA E2 can generate apoptosis in tumoral cells and also can cause a strong immune response directed towards cells that carry the papilloma virus. It has been also found that the virus MVA E2 generates a favorable cellular immune response mediated mainly by macrophages, which depend on antibodies that are capable of destroying cancer cells, which carry DNA of oncogenic papilloma viruses.
The MVA E2 virus also generates an immune response against the tumoral antigens, which causes that the pre-cancer lesions can be eliminated efficiently.
Reasons of using MVA are:
The MVA virus has a deletion of 30,000 bp of DNA compared with the wild type vaccinia, is highly attenuated, expressed foreign genes efficiently and does not show any side effects when used with humans in vaccination protocols.
Background:
Recent advances in recombinant DNA technology have facilitated the isolation of specific genes or of part of them and their transfer to bacteria, yeast, plant cells, animal cells, or to viruses that infect these organisms. The material of the transferred gene (either a native gene or a modified gene) is duplicated and propagates when the viruses duplicate or the transformed cells grow. As a result, the transformed cells have the capacity to synthesize the product that has been codified in the sequences of the transferred gene.
The transfer and expression of genes or portions of them among viruses, eucariotic cells and procariotic cells is possible due to the fact that the DNA of all living organisms is chemically similar and it is composed of the same four nucleotides. The basic differences are based on the nucleotide sequence that appears in the genome of the organisms. The specific nucleotide sequence arranged as codons (three nucleotides) codify for specific sequences of aminoacids. On the other hand the codification between the amino acid sequence and the nucleotide sequence of DNA is essentially the same for all organisms1.
The genomic DNA is organized in sequences that codify for proteins (structural genes) and control regions (these are DNA sequences that control the initiation of the transcription phase and are usually known as promoters) that regulate the expression of structural genes. In general the RNA polymerase is activated in the region of the promoter and transcribes the information along the structural gene to a messenger RNA (mRNA). The mRNA contains recognition sequences, which are the signals for union to the ribosome and signals for the initiation and termination of the protein synthesis (translation). The recent advances on the genetic analysis of the role that play the transcriptional signals in the promoter regions of the genes (that are described usually as the region of 5xe2x80x2 of the genes) have facilitated the possibility of removing and selectively alter the sequence of DNA with the purpose to study its function and its role in the expression of genes and to take certain sequences to study its function in heterologous biological systems such as the system host-vector of recombinant DNA technology1.
The promoters in eucariotic organisms are usually characterized by their content of conserved nucleotides sequences that have structural similarity to the sequences of the promoters of procariotic organisms and this also suggests that they are involved in the regulation of the transcription process. The first of these sequences is rich in adenine and thymine (the position is classified as TATA), which is located 20-30 base pairs up stream from the initiation point of RNA (this site is where the transcription for mRNA initiates) and it is characterized because it contains the consensus sequence (5xe2x80x2TATAA-ATA-3xe2x80x2). The second consensus sequence it is localized 70-90 base pairs up stream and presents the consensus sequence 5xe2x80x2-GG(C/T) CAATCT-3xe2x80x2. All these sequences can be genetically manipulated and be used for the insertion of specific genes. Also these sequences can be altered by other techniques such as directed mutagenesis1.
The restriction enzymes that are used for cloning of genes are specific proteins that cut the nucleic acids in specific sites. Which means that they recognize a sequence (recognition site) in the DNA where to cut.
All cells of an organism cannot synthesize all the proteins; only some of them are present in the cell. This means that the proteins are expressed only in some type of cells, which is the result of the differentiation of cells.
Definitions used in this Document:
The term gene is referred to a sequence of DNA that carries the necessary information for the synthesis of a polypeptide or a protein.
Cloning vehicle is a fragment of DNA that it is normally extra-chromosomal and contains some natural sequences of bacterial genome, to allow its replication inside bacteria. Normally present in a circular form.
The term infection is referred to the entrance of an infectious agent into a cell where if the conditions are favorable it can replicate itself and develop.
The term transfection describes a technique in which we can introduce genetic material into a cell using some salts like calcium chloride of salt or by using liposomes.
The expression systems are those which can synthesize a foreign protein in a bacterial cell.
Systems for the introduction of genes into cells or bacterias2.
Through the use of cloning vectors and with the techniques of transfection, it is possible to introduce genes into a cell and obtain that the genes are efficiently expressed. These vectors can be plasmids or recombinant viruses, which normally are introduced in bacterial systems, mammalian cells, yeast and insect cells. For example the bacteria are expression systems widely used because they can synthesize large quantities of protein at a very low cost, also these systems have been designed to carry on a fast purification of the produced protein. One of the inconveniences about bacteria is that if the protein that it is expressed is unstable inside the cell, it can be degraded rapidly and the yield can go very low. Another disadvantage is that when bacteria are used to express genes from an eucariotic origin (for example genes from mammalian cells) if the protein requires to be modified after its synthesis for its complete biological activity, for example if it needs the addition of carbohydrates (glycosilation) the bacteria is incapable of adding this functional groups that are absolutely essential and so at the end the protein is inactive. Expressing the protein in other systems such as yeast or mammalian cells can solve these products.
Yeast is an excellent expression system for proteins. It is also cheap to grow them and they do not require that the temperature should be maintained at 37xc2x0 C. for its growth; they can grow at room temperature. As in the case of bacteria, the yeasts are not capable of producing all the modifications that some proteins require after its synthesis (post-translational modification).
The viral vectors such as baculovirus (virus that infect insects), are used today for the expression of many proteins, they also have the advantage that the synthesized protein can be transported outside the cell, which greatly facilitates the purification procedure. Nevertheless the insect cells have a small disadvantage of having only two or three systems for glycosylation, which is required by a certain protein to obtain its complete biological activity.
Other viral vectors such as the viruses belonging to the family of the pox virus (for example vaccinia virus) are used today for the production of protein with biological activity because after infecting a mammalian cell, the proteins that are expressed, all of them posses all the necessary modifications to achieve its normal biological activity.
Which is more important and innovative is that the viruses that are constructed (viral expression vector) are used today for vaccine production as well as for therapeutic and preventive treatment against different types of diseases. Also all these viruses are completely safe and they do not cause any secondary adverse reaction, both in animal and in humans, being characterized as vectors of high efficiency for the development of vaccines.